Acoustically mediated fluid transfer methods and uses thereof

ABSTRACT

Acoustic waves are used to transfer small amounts of fluid in a non-contact manner. Acoustic waves are propagated through a pool of a source fluid in such a manner that causes the ejection of a single micro-droplet from the surface of the pool. The droplet is ejected towards a target with sufficient force to provide for contact of the droplet with the target. Because the fluid is not contacted by any fluid transfer device such as a pipette, the opportunities for contamination are minimized. Methods may be employed to transfer fluids from an array of source sites to an array of target sites, thereby enabling the precise automation of a wide variety of procedures including screening and synthesis procedures commonly used in biotechnology.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/735,709, filed on Dec. 12, 2000, the contents of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to non-contact fluid transfer methods,apparatus and uses thereof.

BACKGROUND

Many methods for the precision transfer and handling of fluids are knownand used in a variety of commercial and industrial applications. Thepresently burgeoning industries of biotechnology and biopharmaceuticalsare particularly relevant examples of industries requiring ultra-purefluid handling and transfer techniques. Not only is purity a concern,current biotechnological screening and manufacturing methods alsorequire high throughput to efficiently conduct screening of compoundlibraries, synthesis of screening components, and the like.

Current fluid transfer methods require contacting the fluid with atransfer device, e.g., a pipette, a pin, or the like. Such contactmethods dramatically increase the likelihood of contamination. Manybiotechnology procedures, e.g., polymerase chain reaction (PCR), have asensitivity that results in essentially a zero tolerance forcontamination. Accordingly, a non-contact method for fluid transferwould result in a drastic reduction in opportunities for samplecontamination.

Current biotechnology screening techniques may involve many thousands ofseparate screening operations, with the concomitant need for manythousands of fluid transfer operations in which small volumes of fluidare transferred from a fluid source (e.g., a multi-well platecomprising, for example, a library of test compounds) to a target (e.g.,a site where a test compound is contacted with a defined set ofcomponents). Thus, not only the source, but also the target may comprisethousands of loci that need to be accessed in a rapid,contamination-free manner.

Similarly, biotechnology synthesis methods for the generation of toolsuseful for conducting molecular biology research often require manyiterations of a procedure that must be conducted without contaminationand with precision. For example, oligonucleotides of varying lengths aretools that are commonly employed in molecular biology researchapplications, as, for example, probes, primers, anti-sense strands, andthe like. Traditional synthesis techniques comprise the stepwiseaddition of a single nucleotide at a time to a growing oligomer strand.Contamination of the strand with an erroneously placed nucleotiderenders the oligonucleotide useless. Accordingly, a non-contact methodfor transferring nucleotides to the reaction site of a growing oligomerwould reduce the opportunity for erroneous transfer of an unwantednucleotide that might otherwise contaminate a pipette or othertraditional contact-based transfer device.

Furthermore, existing fluid transfer methods are limited, and do notconveniently and reliably produce the high efficiency, high-densityarrays. Such arrays are also useful in conducting screening, synthesis,and other techniques commonly used in biotechnology.

Accordingly, there exists a need in the art for a non-contact method forthe precision transfer of small amounts of fluid in a rapid manner thatis easily automated to meet industry needs.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the prior art, the presentinvention provides non-contact methods for the transfer of small amountsof fluid. Methods according to the present invention employ the use ofacoustic waves to generate micro-droplets of fluid. In the methods,acoustic waves are propagated through a pool of a source fluid to causethe ejection of at least one, e.g., a single micro-droplet, from thesurface of the pool. The droplet is ejected towards a target withsufficient force to provide for contact of the droplet with the target.

The methods of the invention are easily automated in a manner thatprovides for the processing of many different sources of fluid from anarray of pools of source fluid, and further provides for an array oftarget sites to receive the micro-droplets of source fluid as they areejected from the pools of source fluid. In this manner thousands ofindividual samples of source fluid can be processed and directed to thesame or two or more (e.g., a thousands or more) separate target sitesfor further reaction, detection, and the like. Thus, the presentinvention, because of its non-contact methodology, not only has greaterintrinsic reliability than is provided by presently available liquidejection on demand and continuous stream piezoelectric type pumps, butalso is compatible with a wider variety of liquid compounds, includingliquid compounds which have relatively high viscosity and liquidcompounds which contain particulate components.

The invention provides a non-contact method for transferring smallamounts of source fluid to a target, said method comprising propagatingan acoustic wave from an acoustic liquid deposition emitter through asource fluid containment structure into a pool of source fluid, whereinsaid acoustic liquid deposition emitter is in contact with said sourcefluid containment structure typically through a coupling medium which isinterposed between said acoustic liquid deposition emitter and a firstsurface of said source fluid containment structure, said pool of sourcefluid is on a second surface of said source fluid containment structurethat is opposite or adjacent to said acoustic liquid deposition emitter,and said acoustic wave causes controlled ejection of at least onedroplet of said source fluid from said pool to said target.

The invention also provides a non-contact method for transferring smallamounts of a source fluid to a separate target structure, said methodcomprising activating a piezoelectric transducer thereby propagating anacoustic wave through a coupling medium which is interposed between saidpiezoelectric transducer and a first surface of a source fluidcontainment structure, wherein said source fluid is contained on asecond surface of said source fluid containment structure that isopposite said piezoelectric transducer, and said target is positioned toreceive a droplet of fluid ejected from said source fluid as a result ofpropagation of said acoustic wave through said source fluid.

The invention further provides a method for transferring small amountsof a source fluid from a pool selected from one of a plurality of poolsof source fluid located on a first surface of a source fluid containmentstructure, to a separate target structure without physically contactingsaid source fluid, said method comprising propagating an acoustic wavethrough said source fluid such that a single droplet of fluid is ejectedfrom the surface of said pool of source fluid with sufficient energy tobring said droplet into contact with said target, wherein said acousticwave is propagated from a piezoelectric transducer, said piezoelectrictransducer is in contact, opposite to, or adjacent with said sourcefluid containment structure via a coupling medium interposed betweensaid piezoelectric transducer and a second surface of said source fluidcontainment structure, said second surface of said source fluidcontainment structure is opposite said pool of source fluid, and saidtarget is opposite or adjacent to said surface of said pool of sourcefluid.

The invention also provides an apparatus for performing non-contacttransfer of small amounts of source fluid. The apparatus includes anacoustic liquid deposition emitter and a stage wherein the stage isconfigured to support a source fluid containment structure supportedsuch that the acoustic liquid deposition emitter is in operative contactwith the source fluid containment structure when a coupling medium isinterposed there between. The apparatus may include a number ofadditional elements, including, for example: an acoustic wave channelstructure that is mechanically coupled to the acoustic liquid depositionemitter (e.g., a piezoelectric transducer) to provide for transmissionof an acoustic wave from, e.g., the piezoelectric transducer to saidcoupling medium; a structure for maintaining the coupling medium inoperative contact with the acoustic liquid deposition emitter; a lensfor focusing said acoustic wave; controls for varying one or more offrequency, voltage, and duration of an energy source used to excite theacoustic liquid deposition emitter and thereby propagate an acousticwave; a stage actuator for user-defined positioning of the stagerelative to the acoustic liquid deposition emitter; a focussing actuatorfor user-defined positioning of said acoustic liquid deposition emitterrelative to said stage; a computer for controlling the stage actuatorand/or the focussing actuator; and a fluid level detector for detectinga level of fluid in a source fluid containment structure supported bysaid stage.

The invention also provides a system for performing non-contact transferof small amounts of a source fluid. The system includes a source fluidcontainment structure, a movable stage configured to support the sourcefluid containment structure, an acoustic liquid deposition emitter inoperative contact with the source fluid containment structure, acoupling medium interposed between the deposition emitter and the sourcefluid containment structure, and a computer in operable communicationwith the acoustic liquid deposition emitter for varying one or more offrequency, voltage and duration of an energy source used to excite theacoustic liquid deposition emitter and wherein the computer is incommunication with the movable stage for positioning the source fluidsuch that operative contact with the acoustic liquid deposition emitter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating one embodiment of anon-contact fluid transfer apparatus of the present invention.

FIG. 2 is a schematic diagram illustrating one embodiment of the presentinvention, where an acoustic wave 10 generated by a piezoelectricelement 60 is propagated through a wave channel 70, a coupling medium 20and a source fluid containment structure 30 to a pool of source fluid40, causing ejection of a droplet 50 of source fluid from the surface ofthe pool.

FIG. 3 is a schematic diagram illustrating an embodiment of the presentinvention where each pool of source fluid 40 is confined by a coating ofa hydrophobic material 80 on source fluid containment structure 30.

FIG. 4 depicts a lens contemplated for use in the practice of thepresent invention, and shows various parameters that may be adjusted toprovide correct focus of an acoustic wave. The diameter of the apertureis 2 a, Z₀ is the focal length, d_(z) is the depth of field, and d_(r)is the deposition feature diameter.

FIG. 5 is a schematic diagram illustrating several options formonitoring source fluid pool levels by monitoring acoustic wavesgenerated by secondary piezoelectric elements 65 directed at the sourcefluid pool.

FIG. 6 is a schematic diagram illustrating an embodiment of the presentinvention where a computer 100 receives signals generated by a secondarypiezoelectric element 65 and computes source fluid pool 105 levels fromthe information received. The computer of 100 then compares 110 thecomputed height versus the emitter position 115 and controls the focusof the primary piezoelectric element by moving the emitter (60 of FIG.2) relative to position the source fluid pool (40 of FIG. 2) usingpositioning stage 120 to most effectively eject droplet(s) from thesurface of the source fluid pool (40 of FIG. 2).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided an apparatususeful for non-contact fluid transfer. With reference to FIG. 1 there isschematically presented one embodiment of an apparatus of the presentinvention. The figure depicts a non-contact fluid transfer apparatus 5having at least one acoustic liquid deposition emitter 60 in electricalcommunication with a computer 95. During operation the acoustic liquiddeposition emitter 60 generates an acoustic wave or beam 10 that can bepropagated through an optional wave channel 70. The acoustic wave can befocused by tens 75 prior to propagating through coupling fluid 20 tooptimize the energy of the acoustic wave or beam 10 upon the liquid/airinterface of source fluid 40. The acoustic wave 10 is propagated througha coupling medium 20 after which the wave is transmitted through sourcefluid containment structure 30 where the wave comes to focus at or nearthe surface of a pool of source fluid 40 thereby causing ejection of atleast one droplet 50 of source fluid from the surface of the pool. Inone embodiment, the ejected droplet 50 makes contact with a target 80.The source fluid containment structure 30 can be held on a movable stage35. The movable stage 35 is controlled by actuator mechanism 85 whichcontains a horizontal actuator 85′ or a vertical actuator 85″ or acombination of the two actuators to control the movement of the stage 35in both the vertical and horizontal directions. The actuator 85 istypically in communication with computer 95 which controls the movementof the stage to select a source fluid 40 or to adjust focusing of theacoustic wave or beam 10 upon the source fluid 40. The computer may haveimplemented thereon various algorithms to adjust the focal length andenergy of the acoustic deposition emitter as well as control and managethe location of the acoustic deposition emitter relative to a particularsource fluid present in or on a source fluid containment structure.

In accordance with the present invention, there are provided non-contactmethods for transferring small amounts of source fluid to a target. Themethods of the invention comprise propagating an acoustic wave from anacoustic liquid deposition emitter through a source fluid containmentstructure into a pool of source fluid. The acoustic liquid depositionemitter is coupled with the source fluid containment structure typicallythrough a coupling medium, which is interposed between the acousticliquid deposition emitter and a first surface of the source fluidcontainment structure. The pool of source fluid is on a second surfaceof the source fluid containment structure, and the second surface isopposite the first surface, which is in contact with the couplingmedium. Thus, the acoustic wave is emitted from the acoustic liquiddeposition emitter, propagates through the coupling medium, across orthrough the source fluid containment structure to cause controlledejection of at least one droplet of the source fluid from the pool tothe target. By “at least one droplet” means one or more droplets or aplurality of droplets. The droplets can be ejected substantiallysimultaneously or sequentially. In preferred embodiments a singleindividual droplet is ejected using the methods of the invention.

For an example of one embodiment of the present invention, reference ismade to FIG. 2 which shows the propagation of an acoustic wave 10through a coupling medium 20 after which the wave is transmitted throughsource fluid containment structure 30 where the wave comes to focus ator near the surface of a pool of source fluid 40 thereby causingejection of at least one droplet 50 of source fluid from the surface ofthe pool.

As used in the context of the coupling medium, “coupled with” or“coupled to” means that the coupling medium provides a medium for theacoustic waves to travel between the acoustic liquid deposition emitterand the fluid containment structure. In a preferred embodiment, thecoupling medium is in contact with both the acoustic liquid depositionemitter and a first surface of the fluid containment structure (e.g.,the underside of the structure, if the fluid containment structure isoriented with source fluid on its top surface).

As used herein, “controlled ejection” means that the acoustic wave canbe adjusted, as further described herein, to vary the size and/or numberof droplets ejected from the surface of the pool. Such controlledejection techniques can involve adjusting or focusing of the acousticwave, frequency of the acoustic wave, modifying the distance between theacoustic liquid deposition emitter and the source fluid, and the like,in response to the type of source fluid (e.g., the source fluidscontent, viscosity and the like) as well as changes in the volume orlevel of the source fluid during ejection of droplets or as a result ofevaporation.

Accordingly, the methods of the invention rely on the fact that anacoustic wave may be propagated through a pool of fluid in a manner thatcauses ejection of a single droplet of fluid from the surface of thepool of fluid to assist in transferring the fluid from the source poolto a desired target. Because the acoustic wave is of sufficient energyand properly focused to eject at least one droplet of fluid from thesurface of the source pool, no pipettes or other fluid handling devicesneed come into contact with the source pool of fluid.

Any type of fluid is suitable for use in the practice of the presentinvention. As used herein, “fluid” means an aggregate of matter in whichthe molecules are able to flow past each other without limit and withoutthe formation of fracture planes. Thus, as recognized by those of skillin the art, a fluid may comprise a liquid and/or a gas under theappropriate conditions. A fluid may be homogenous, i.e., one component,or heterogeneous, i.e., more than one component. Where a plurality ofpools of source fluid are employed in the practice of the presentinvention, each pool may comprise a different source fluid, as furtherdescribed herein.

“Non-contact,” as used herein, means that a source fluid is transferredor removed from a source pool of fluid without contacting the sourcefluid with a transfer device. In one embodiment, the source fluid istransferred from a source pool of fluid to a target without contactingthe source fluid with a transfer device. For example, the ejection of adroplet of source fluid from the source fluid containment structure doesnot contact anything other than the target towards which the droplet isdirected. Thus, no pipettes, pins, capillaries or other fluid transferdevices are brought into contact with the source fluid. In this manner,opportunities for contamination of the fluid and the system areminimized. Moreover, non-contact fluid transfer as described herein doesnot require the use of nozzles with small ejection orifices that easilyclog. In addition, the relatively high cost piezoelectric transducersand acoustic focusing lenses remain as fixed components of the fluiddelivery/transfer system, while the fluid(s) being transferred, as wellas the fluid containment structure, may constitute separate anddisposable components. This allows for a greatly improved cost ofownership because the relatively high cost piezoelectric transducer(s)and acoustic focusing lens(es) never contact the individual fluidcompounds.

As used herein, “source fluid containment structure” is any structuresuitable for containing or supporting a pool of source fluid and whichallows an acoustic wave to propagate from a first side or end of thestructure, through the structure to the second side or end of thestructure, wherein the source fluid is contained on the second side orwithin the structure. Thus, suitable source fluid containment structuresinclude a flat structure such as a slide (e.g., a glass or polystyrenemicroscope slide), or the like, onto which one or more discrete pools ofsource fluid may be deposited; also included are single and multi-wellplates commonly used in molecular biology applications; capillaries(e.g., capillary arrays); and the like.

Maintaining discrete pools of source fluid may be accomplished by avariety of methods, including providing a plurality of separationstructures such as wells, tubes or other devices that have at least onewall separating one fluid from another, or by providing coatings thatserve to define containment regions (as further described herein), andthe like. Immiscible fluids or materials can be used to separatedissimilar fluids (e.g., waxy coatings separating hydrophilic fluids)forming containment fields.

Source fluid containment structures may be constructed of any suitablematerial, bearing in mind the need for good acoustic velocityproperties. Such materials include glass, polymer (e.g., polystyrene),metal, a textured material, a containment field, and the like, as wellas combinations thereof. The material may further be porous ornon-porous, or combinations thereof.

Source fluid containment structures may have one or more coatings tofacilitate fluid containment. Thus, in one embodiment of the presentinvention, slides having zones of relative hydrophobicity andhydrophilicity may be employed as source fluid containment structures.In this manner, an aqueous fluid may be applied to a zone of the slidethat is surrounded by a relatively hydrophobic region (or coating ofrelatively hydrophilic material), thereby operating to contain a pool ofsource fluid. Reference is made to FIG. 3 as one example of thisembodiment of the present invention. Source fluid containment structure30 has zones or regions (i.e., containment zones) for containingdiscrete pools of source fluid 40. These zones are defined by a coatingof a hydrophobic material 80 which acts to confine the source fluid 40in the containment zones. Accordingly, in this embodiment of the presentinvention, sample wells are not required to contain the discrete poolsof source fluid.

Examples of hydrophobic coatings include polytetratfluoroethylene(PTFE), hydrophobic amino acids, polypeptides comprising hydrophobicamino acids, waxes, oils, fatty acids, and the like. Those of skill inthe art can readily determine a number of other hydrophobic coatings,which may also serve to define source fluid containment zones, andcontain source fluids therein. Optionally, the zone(s) of the slidewhich are chosen to contain non-aqueous source fluid may have relativelyhydrophilic regions (or coating of relatively hydrophilic material) tofurther define the containment zone(s). Thus, pools of source fluid canbe confined to defined areas of a slide by virtue of the relative areasof hydrophobicity and hydrophilicity. Again, sample wells are notrequired to contain a pool of source fluid.

The methods of the invention are contemplated for use in high throughputoperations. It is preferred that the source fluid containment structurehave multiple containment regions, preferably in an array which can bemapped so that each containment region can be accessed under directionof a controlling computer. Thus, in one preferred embodiment of thepresent invention, the source fluid containment structure is amulti-well plate such as a micro-titer plate (comprising a plurality ofwells, each having a bottom, sides and an open top for the ejection of adroplet there through). Suitable micro-titer plates may have from about96 to about 1500 wells, or more. One example of a suitable plate is a1536 well plate (e.g., catalog number 3950 available from CorningCorporation).

As used herein, “target” means a structure or a zone towards which adroplet of source fluid is ejected, or with which the ejected dropletmakes contact. The target may be constructed of any material that issuitable for receiving the ejected fluid droplet, including, forexample, a glass, a polymer, a paper, a gel, a conductive material, ametal, a porous material, a non-porous material, a textured material, orthe like. The material may be further coated or textured to receive andretain the droplet of fluid. Coatings contemplated for use in thepractice of the present invention include polytetrafluoroethylene(PTFE), aminomethylated or highly crosslinkedpolystyrene-divinyl-benzene, and the like. In some embodiments of thepresent invention, it may be desirable to direct a fluid droplet to ameasuring device or other remotely located zone, thus, the target maynot comprise a tangible object but instead comprise a collection zonedefined by a containment field, a conduit, a chamber, a collector, acontainer, or the like. In this manner, a droplet of fluid could bedirected, for example, to a conduit that leads to the reaction chamberof a mass spectrometer, or the like.

In one embodiment, the target is separate in that it is not in contactwith the source fluid containment structure, but rather can be held inplace at a selected distance from the source containment structure. Ofcourse the distance must be within the effective range of the dropletgenerated by the acoustic liquid deposition emitter. The droplets of thesize ejected from the source pool are small (e.g., at least about 1micrometer), that in a vacuum they travel a relatively large distance(i.e., many centimeters) in opposition to the force of gravity. One ofskill in the art will recognize that the distance said ejected sourcematerial can travel will depend upon the size and content of the ejectedfluid and the surrounding atmospheric humidity, temperature and thelike. In addition, the properties of the acoustic wave (e.g., frequencyand the like) generated by the liquid deposition emitter can be variedto adjust the distance and size of the ejected source fluid droplet. Theformation of a given droplet is thus dependent on, for example, thefrequency of the liquid deposition emitter's (e.g., a piezoelectrictransducer's) oscillation. Accordingly, a target (in still air)positioned about two (2) centimeters above the surface of the sourcepool can easily be impacted with a droplet ejected from the surface ofthe source pool. Thus, while a distance in excess of a millimeter can beemployed in the practice of the present invention, it is presentlypreferred that the target be positioned no more than about 0.25millimeter from the surface of the source pool; and in a anotherpreferred embodiment, the target is no more than about five (5)millimeters from the surface of the source pool.

As used herein, “acoustic deposition emitter” means any device capableof generating a directional acoustic wave capable of causing ejection ofat least one droplet of fluid from the surface of a pool of fluid. Asunderstood by those of skill in the art, an acoustic wave or beam exertsa radiation pressure against objects upon which it impinges. Thus, whenan acoustic wave or beam impinges on a free surface (e.g., fluid/airinterface) of a pool of fluid from beneath, the radiation pressure whichit exerts against the surface of the pool may reach a sufficiently highlevel to release at least one individual droplet of fluid from the pool,despite the restraining force of surface tension. In a preferredembodiment, a piezoelectric transducer is employed as an acousticdeposition emitter. In one embodiment, a piezoelectric transducercomprises a flat thin piezoelectric element, which is constructedbetween a pair of thin film electrode plates. As is understood by thoseof skill in the art, when a high frequency and appropriate magnitudevoltage is applied across the thin film electrode plates of apiezoelectric transducer, RF energy will cause the piezoelectric elementto be excited into a thickness mode oscillation. The resultantoscillation of the piezoelectric element generates a slightly divergingacoustic beam of acoustic waves. By directing the wave or beam onto anappropriate lens having a defined radius of curvature (e.g., a sphericallens, or the like), the acoustic beam can be brought to focus at adesired point.

The radiation pressure is greatest in the acoustic wave or beam's focalregion, particularly, at the pool surface where wave reflection occurs.The pressure caused by the acoustic wave or beam acts to lift a smallcolumn of liquid which appears initially as a small mound. When enoughenergy is applied to overcome surface tension the mound becomes amomentary liquid fountain where each tone burst emits a single droplet.Because the focused wave or beam is diffraction limited, the dropletdiameter is proportional to the wavelength. Observations with waterindicate that single droplet ejection occurs at a specific power levelband where uniformly sized droplets form. However above this band, asone increases power level further the droplets begin to form tails whichthen break off into satellite droplets. Further increases in powercauses the process to transition to a continuous fountain.

At energy levels just below the threshold of normal droplet ejection, afine mist may be emitted from the source fluid. The mist may be used insituations where it is desirable to coat a surface with fine dropletcoating that is 1/10 to 1/100 the size of the normally produceddroplets.

Fountain ejection can be achieved when the power level is well beyondthe normal single droplet ejection range. Fountains appear to becontinuous or nearly continuous streams of liquid that eject and breakup in a random fashion and produce widely distributed sizes. This modemay be used for producing a spray like coating.

In addition, it is possible to affect the trajectory of the ejecteddroplet by means of electrostatics. The same principals are used in thecommon cathode ray tube. A simple charging plate positioned parallel tothe pool surface is used. The pool acts as an opposing plate similar toa capacitor. Therefore, the pool will acquire charge that is oppositethat of the charging plate. When a droplet is ejected it carries aisolated charge at point where it breaks off the pool. A small diameterhole in the charging plate permits droplet charging without impeding itspath. There is an acceleration experienced by the droplet so that itsfinal velocity will be the combination of initial ejection velocity andan electrostatic acceleration. The charge electrode voltage may bemanipulated to accelerate droplets if higher velocity is desired.

Deflection is accomplished in a manner identical to the cathode raytube. The deflection plates set up an electric field perpendicular tothe droplets flight path. An acceleration perpendicular to the pathresults in a deflected trajectory. By manipulating the deflectionvoltage in two axes a sweep pattern is formed.

Accordingly, to eject individual droplets from the source fluidcontainment structure on demand, the RF excitation of the piezoelectricelement is amplitude or frequency modulated (by means well understood tothose of skill in the art), thereby causing the focused acoustic beamradiation pressure exerted against the surface of the source pool offluid to swing above and below a predetermined droplet ejectionthreshold level. Thus, the RF voltage applied to the piezoelectricelement may be amplitude or frequency modulated and/or energy durationmodulated to control the droplet ejection process. In a preferredembodiment, the RF excitation voltage is computer controlled and may bechanged to account for changes in the viscosity and surface tension ofthe source fluid.

In one embodiment, a computer sends an analog voltage pulse to thepiezoelectric transducer by an electrical wire. The voltage pulse can becontrolled, for example, by a MD-E-201 Drive Electronics manufactured byMicrodrop, GmbH, Muhlenweg 143, D-22844 Norderstedt, Germany. Theelectronics can thus control the magnitude and duration of the analogvoltage pulses, and also the frequency at which the pulses are sent tothe piezoelectric transducer. Each voltage pulse causes the generationof an acoustic wave from the piezoelectric transducer, which in turn ispropagated through a coupling medium and into or through the sourcefluid thereby impinging on the surface of the source fluid. For example,an acoustic wave (e.g., a pressure wave) propagates through the couplingmedium and source fluid where one droplet of source fluid is emittedunder high acceleration. The size of these droplets has been shown to bevery reproducible. The high acceleration of the source fluid minimizesor eliminates problems caused by source fluid surface tension andviscosity, allowing extremely small droplets to be expelled from thesurface of a pool of source fluid, e.g., as small as 5 picoliterdroplets have been demonstrated.

The piezoelectric transducer may employ a flat crystal disk, or othercrystal designs, e.g., square, perforated disk, and the like. In apresently preferred embodiment, the piezoelectric transducer is a flatdisk. Because most electronic circuits are designed for a 50Ω (ohm)load, it is presently preferred to employ a 50Ω (ohm) transducer. Whileany material may be used in the piezoelectric element, in a presentlypreferred embodiment of the invention, a Navy Type I piezoelectricmaterial is employed in a disk element having diameter D=0.039 inch orD=0.991 mm. Other shapes of piezoelectric crystals are also contemplatedfor use in the practice of the present invention.

Firing of the acoustic deposition emitter may be conducted manually orunder direction of a controlling computer. Because the present inventionis useful in high throughput operations, it is presently preferred thatfiring of the acoustic deposition emitter be computer controlled. Firingof the emitter can be coordinated with computer controlled positioningof both the source containment structure or the target so that aspecific source fluid can be directed to a specifically selected targetspot on the target.

Proper focus of the acoustic wave can be achieved by providing a lensbetween the piezoelectric transducer and the coupling medium. Lensescontemplated for use in the practice of the present invention may be ofconstant curvature or aspherical. An aspherical lens (i.e., a lenshaving a compound curvature) may be employed to accommodate anyirregularities in the acoustic wave, whether due to the piezoelectricelement itself, a misalignment of the piezoelectric element with thesurface of the pool of source fluid, or the like.

To capture the maximum amount of energy emitted by the crystal, it ispreferred that the lens aperture be greater than the crystal diameter.With reference to FIG. 4, the lens can be constructed with a sphericalcutter, for example, to have a selected focal distance Z_(O). It ispreferred that Z_(O)=0.125 inch or 3.175 mm. This yields an f-value(f=Z_(O)/D) equal to four (4), where D is the diameter of the activearea of the piezoelectric material. It is preferred that the radius ofcurvature of the lens be chosen to provide an f-value in the range ofabout 1 to 4. In another aspect of this embodiment, the f-value is inthe range of 1-2. In yet another aspect of this embodiment, the f-valueis in the range of 2-4.

To efficiently capture the energy in the acoustic wave generated by thepiezoelectric crystal, it is desirable that the diameter of the lens begreater than the diameter of the active portion of the piezoelectriccrystal. Thus, in view of the preferred active crystal diameter of 0.039inches or 0.99 mm, the presently preferred value for the radius of thelens (a) is about 0.016 inch or 0.40 mm (see FIG. 4). In a typicalembodiment, the focal distance of the lens may be approximately equal to2.5 to 3 times the diameter of the crystal.

By virtue of having an f-value in the range of 1-4, a relatively longfocal length (d_(z)) results. Consequently, the acoustic depositionemitter is functional over a wide range of depths of source pool. Inthis manner, refocusing of the emitter is not required every time thedepth of a particular sample pool is altered by the ejection of somematerial therefrom. Nonetheless, in an alternative embodiment of thepresent invention, adjusting the focus of the acoustic beam iscontemplated. Such adjustment may be made by varying the distancebetween the acoustic deposition emitter and the surface of the pool ofsource fluid. Any methods useful for varying the distance between theacoustic deposition emitter and the surface of the pool of source fluidare contemplated for use in the practice of the present invention.Focussing may be automated and controlled by computer.

By applying a particular wavelength (λ) of the acoustic wave in thesource fluid, the depth of focus can be estimated by applying theformula dz=4.88·λ·f². The wavelength (λ) of the acoustic wave can bedetermined by those of skill in the art based on the velocity of soundthrough the chosen source fluid and the frequency of the acoustic wave.Thus, when the source fluid comprises water, the relevant equations are${V_{H\quad 2O} = {1496\quad m\text{/}s}},{{{and}\quad\lambda} = \frac{VH2O}{frequency}}$

Droplet diameter (d_(r)) at a given λ and f-value can be determined byapplying the equation d_(r)=1.02·λ·f. Similarly, a selected dropletdiameter can be achieved by solving the preceding equation for λ, andemploying acoustic waves of that wavelength.

By applying the forgoing equations to the preferred values for variables(f) and (a) disclosed herein, and assuming a source fluid comprisingwater, the wavelength λ=75 μm; the focal length d_(z)=3.75 mm; and thedroplet diameter d_(r)=245 μm.

The size of the droplet can also be adjusted by modulating one or moreof frequency, voltage, and duration of the energy source used to excitethe acoustic liquid deposition emitter (e.g., a piezoelectrictransducer). Accordingly, a wide range of user-defined droplet diameterscan be achieved by employing the methods of the invention. In oneembodiment of the present invention, the defined droplet diameter is atleast about 1 micrometer. In another embodiment of the presentinvention, the defined droplet diameter is in the range of about 1micrometer to about 10,000 micrometers. In yet another embodiment of thepresent invention, the defined droplet diameter is in the range of about500 micrometers to about 1000 micrometers. In a further embodiment ofthe present invention, the defined droplet diameter is in the range ofabout 60 micrometers to about 500 micrometers. In yet another embodimentof the present invention, the defined droplet diameter is in the rangeof about 100 micrometers to about 500 micrometers. In another embodimentof the present invention, the defined droplet diameter is in the rangeof about 120 micrometers to about 250 micrometers. In a furtherembodiment of the present invention, the defined droplet diameter is inthe range of about 30 micrometers to about 60 micrometers. In stillanother embodiment of the present invention, the defined dropletdiameter is about 50 micrometers.

It is preferred that acoustic waves be channeled from the liquiddeposition emitter (e.g., piezoelectric element) to the source fluid viaan acoustic wave channel. Reference is made to FIG. 2 which shows anacoustic wave 10 being generated by a piezoelectric element 60 andpropagated through acoustic wave channel 70. The rapid oscillation ofthe piezoelectric element 60 generates an acoustic wave 10, whichpropagates through the acoustic wave channel 70 at a relatively highvelocity until it strikes the focusing lens 75. The wave then emergesinto a medium 20 (i.e., the coupling medium) having a much loweracoustic velocity, so the spherical shape of the lens imparts aspherical wave-front to it, thereby forming the acoustic beam. Theacoustic wave channel 70 may be constructed of aluminum, silicon,silicon nitride, silicon carbide, sapphire, fused quartz, certainglasses, or the like. In a preferred embodiment, the acoustic wavechannel 70 is constructed of aluminum. Each of the aforementionedmaterials is chosen because of its high acoustic velocity typeproperties. In general, suitable materials have an acoustic velocity,which is higher than the acoustic velocity of the source fluid. It isalso preferred that the piezoelectric element 60 is deposited on orotherwise intimately mechanically coupled to a surface of the acousticwave channel 70.

In a preferred embodiment, a sufficiently high refractive index ratio ismaintained between the acoustic wave channel and the source containmentstructure by providing a temperature controlled liquid transitioninterface (e.g., a temperature controlled coupling medium as describedherein) that couples the highly focused acoustic wave or beam with acontainment structure. The focusing lens should direct the beam into anessentially diffraction limited focus at or near the fluid/air interfaceat the surface of the source fluid pool.

As used herein “coupling medium” means a fluid medium having an acousticimpedance that is substantially the same as the acoustic impedance ofthe source fluid containment structure. The coupling medium will be incontact with both an acoustic liquid deposition emitter or preferablythe acoustic wave channel and one side of the fluid containmentstructure, thereby providing for efficient energy transfer from theacoustic wave channel to the fluid containment structure, andsubsequently through the source fluid. As an example, a polystyrenemulti-well plate has an acoustic impedance of about 2.3. Water has anacoustic impedance of about 1.7. Accordingly, water is a good couplingmedium when the source fluid containment structure is a polystyrenedevice (e.g., a multi-well plate) due the close match in impedancevalues between water and the plate. By adding other fluids (e.g.,glycerol, or the like) to the water, an even closer match can beachieved. Other fluids may also be employed in the practice of thepresent invention.

Thus, by providing a coupling medium between the acoustic wavedeposition emitter or preferably the acoustic wave channel and the fluidcontainment structure, a far more efficient transfer of energy occursthan if no coupling medium is employed. In one aspect of the invention,the coupling medium is temperature controlled to minimize any effect oftemperature on the source fluid.

Because these methods may be employed in high throughput applications,it is preferred that methods of the invention further compriseuser-defined positioning of the acoustic liquid deposition emitterrelative to an array of source wells, thus providing for user-definedassociation of the acoustic liquid deposition emitter with a selectedpool of source fluid for ejection of a droplet therefrom. This can beaccomplished by a variety of methods. For example, in the case where amulti-well plate is employed as the source fluid containment structure,a computer-controlled translator (e.g., an actuator, or the like) canmanipulate the position of the multi-well plate or a movable stage uponwhich the multiwell plate rests. Thus, a selected well or a selectedsuccession of wells is placed over the acoustic deposition emitter, asthe source fluid contained in each well is needed for the applicationbeing conducted (e.g., oligonucleotide synthesis, or the like). In arelated embodiment, the acoustic deposition emitter may be moved ratherthan the source plate. For example, the source fluid containmentstructure may remain fixed in position and the acoustic liquiddeposition emitter may be moved relative to a well or particular sourcefluid of interest contained in or on the source fluid containmentstructure. In yet another embodiment, multiple deposition emitters maybe utilized each associated, for example, with a source fluid pool. Inthis embodiment, neither the source fluid containment structure nor thedeposition emitter are moved but rather the deposition emitters areselectively activated depending upon which source fluid is desired tohave at least one droplet ejected there from. Once again, this allowsfor the selective association of the emitter with a selected sourcepool. Accordingly, a source fluid array having a plurality of differentsource fluid materials may have droplets selectively ejected from aparticular source fluid towards, for example, a target.

The target may comprise an array of target zones or target spots towhich source fluid is directed. As described above, with respect to thesource fluid and acoustic deposition emitter, the target may also bemoveable relative to a source fluid. For example, the target may bemoved relative to a source fluid to be ejected thereby allowing forselected receipt at the target of a desired ejected source fluiddroplet. The target may be positioned so that each target zone can beselectively positioned over the selected pool of source fluid. Acomputer controlled actuator arm, or the like can accomplish positioningof the target. It is presently preferred that both the target and thesource fluid containment structure be positionable via separatecomputer-controlled actuators. Thus, the non-contact fluidtransfer/deposition technology described herein provides for precisetargeting of individual source fluids to selected target zones.

Source fluids contemplated for use in the practice of the presentinvention may comprise one or more source materials. Source materialsmay include both biological and chemical compounds, agents and lifeforms (e.g., plant cells, eukaryotic or prokaryotic cells).

As used herein, “biological compounds” may comprise nucleic acids (e.g.,polynucleotides), peptides and polypeptides (including antibodies andfragments of antibodies), carbohydrates (e.g., oligosaccharides), andcombinations thereof. In some embodiments, cells (e.g., eukaryotic orprokaryotic) may be contained in the fluid. Such an embodiment wouldallow for the transfer of organisms from one source fluid to anotherfluid or target during cell culturing or sorting.

The term “polynucleotides” and “oligonucleotides” include two or morenucleotide bases (e.g., deoxyribonucleic acids or ribonucleic acids)linked by a phosphodiester bond. Accordingly, such polynucleotides andoligonucleotides include DNA, cDNA and RNA sequences. Polynucleotidesand oligonucleotides may comprise nucleotide analogs, substitutednucleotides, and the like. Nucleic acids contemplated for use in thepractice of the present invention include naked DNA, naked RNA, nakedplasmid DNA, either supercoiled or linear, and encapsulated DNA or RNA(e.g., in liposomes, microspheres, or the like). As will be understoodby those of skill in the art, particles mixed with plasmid so as to“condense” the DNA molecule may also be employed.

Polypeptides contemplated for use in the practice of the presentinvention includes two or more amino acids joined to one another bypeptide bonds. Thus, polypeptides include proteins (e.g., enzymes (e.g.,DNA polymerase), structural proteins (e.g., keratin), antibodies,fragments thereof, and the like), prions, and the like.

“Chemical compounds” contemplated for use in the practice of the presentinvention may comprise any compound that does not fall under thedefinition of biological compounds as used herein. Specific chemicalcompounds contemplated for use in the practice of the present inventionincludes dyes, detectable labels, non-enzyme chemical reagents,dilutents, and the like.

As used herein, the terms “detectable label”, “indicating group”,“indicating label” and grammatical variations thereof refer to singleatoms and molecules that are either directly or indirectly involved inthe production of a detectable signal. Any label or indicating agent canbe linked to or incorporated in a nucleic acid, a polypeptide,polypeptide fragment, antibody molecule or fragment thereof and thelike. These atoms or molecules can be used alone or in conjunction withadditional reagents. Such labels are themselves well known in the art.

The detectable label can be a fluorescent-labeling agent that chemicallybinds to proteins without denaturation to form a fluorochrome (dye) thatis a useful immunofluorescent tracer. Suitable fluorescent labelingagents are fluorochromes such as fluorescein isocyanate (FIC),fluorescein isothiocyanate (FITC),5-dimethylamine-1-naphthalenesulfonylchloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB-200-SC), and the like. A description ofimmunofluorescence analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody as a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

The detectable label may be an enzyme, such as horseradish peroxidase(HRP), glucose oxidase, and the like. In such cases where the principalindicating label is an enzyme, additional reagents are required for theproduction of a visible signal. Such additional reagents for HRP includehydrogen peroxide and an oxidation dye precursor such asdiaminobenzidine. An additional reagent useful with glucose oxidase is2,2′-azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

In another embodiment, radioactive elements are employed as labelingagents. An exemplary radiolabeling agent is a radioactive element thatproduces gamma ray emissions, positron emissions, or beta emissions.Elements that emit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁶I, ¹³¹I and ⁵¹Cr,represent one class of radioactive element indicating groups. Betaemitters include ³²P, ¹¹¹Indium, ³H and the like.

The linking of a label to a substrate (e.g., labeling of nucleic acids,antibodies, polypeptides, proteins, and the like), is well known in theart. For instance, antibody molecules can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Methods of Enzymology, 73:3-46(1981). Conventional means of protein conjugation or coupling byactivated functional groups are particularly applicable. See, forexample, Aurameas et al., Scandinavia Journal of Immunology. Vol. 8,Suppl. 7:7-23 (1978), Rodwell et al, Biotech., 3:889-894 (1984), andU.S. Pat. No. 4,493,795.

In one embodiment, the methods of the present invention may be used topair certain ligands (i.e., a molecular group that binds to anotherentity to form a larger more complex entity) and binding partners forsuch ligands. For example, certain biological molecules are known tointeract and bind to other molecules in a very specific manner.Essentially any molecules having a high binding specificity or affinityfor each other can be considered a ligand/binding partner pair, e.g., avitamin binding to a protein, a hormone binding to a cell-surfacereceptor, a drug binding to a cell-surface receptor, a glycoproteinserving to identify a particular cell to its neighbors, an antibody(e.g., IgG-class) binding to an antigenic determinant, anoligonucleotide sequence binding to its complementary fragment of RNA orDNA, and the like.

Such pairings are useful in screening techniques, synthesis techniques,and the like. Accordingly, in one embodiment of the present invention,screening assays may be performed in which the binding specificity ofone compound for another is sought to be determined. For example,multiple test compounds (i.e., putative ligands, optionally havingdetectable labels attached) may be screened for specific interactionwith a selected binding partner. Such assays may be carried out bypositioning one of a plurality of putative ligands in each pool of anarray of source fluids. The target may comprise, for example, an arrayof target zones, each zone having affixed to it a sample of the bindingpartner for which specific binding is sought to be identified. Employingthe methods of the invention, a droplet of each putative ligand can beejected to a target zone and the target thereafter washed under definedconditions. Afterwards, each of the target zones is inspected todetermine whether binding of the putative ligand has occurred. Bindingof a putative ligand serves to identify that compound as a ligand forthe binding partner. Binding can easily be identified by any methodknown to those of skill in the art. By employing detectable labeled testcompounds, binding can readily be determined by identifying a labeledcompound bound to the target. Of course, such assays may be reversed,i.e., the selected binding partner may be used as a labeled sourcecompound, while putative ligands are arrayed onto the target.

In one aspect of the foregoing embodiment, the methods of the inventionmay also be applied to the identification of peptides or peptidemimetics that bind biologically important receptors. In this aspect, aplurality of peptides of known sequence can be applied to a target toform an array using methods described herein. The resulting array ofpeptides can then be used in binding assays with selected receptors (orother binding partners) to screen for peptide mimetics of receptoragonists and antagonists. Thus, the invention provides a method forproducing peptide arrays on a target, and methods of using such peptidearrays to screen for peptide mimetics of receptor agonists andantagonists.

The specific binding properties of binding partners to ligands haveimplications for many fields. For example, the strong binding affinityof antibodies for specific antigenic determinants is critical to thefield of immunodiagnostics. Additionally, pharmaceutical drug discovery,in many cases, involves discovering novel drugs having desirablepatterns of specificity for naturally occurring receptors or otherbiologically important binding partners. Many other areas of researchexist in which the selective interaction of binding partners for ligandsis important and are readily apparent to those skilled in the art.

The methods of the invention may also be employed in synthesisreactions. For example, in another embodiment of the present invention,employing monomeric and/or multimeric nucleotides as source compoundscan be employed to synthesize oligonucleotides (useful as probes,labels, primers, anti-sense molecules, and the like). Such sourcecompounds may be present in a fluid medium (i.e., source fluid) and eachsource fluid placed in a defined position of an array on the sourcecontainment structure. By ejecting source nucleotides from the sourcecontainment structure onto a defined target zone of the target, definednucleotides can be added to a growing product oligonucleotide chain inan additive manner that serves to define the nucleotide sequence of thegrowing product oligonucleotide.

The particular chemical reactions necessary to perform oligonucleotidesynthesis are well known to those of skill in the art. Such reactions,or others, which may become known, can be performed in situ on thetarget by, for example, contacting the growing oligonucleotide with thenecessary reagents between each iterative addition of furthernucleotide(s). Flowing the reagents across the target, by passing thetarget through a reagent bath, or the like can perform reagentcontacting. By employing a target with a suitable coating or havingsuitable surface properties, the growing oligonucleotide can be bound tothe target with sufficient strength to undergo the necessary chemicalreactions, after which the mature oligonucleotide can be released fromthe target. For example, methods for attaching oligonucleotides to glassplates in a manner suitable for oligonucleotide synthesis are known inthe art. Southern, Chem. abst. 113; 152979r (1990), incorporated byreference herein in its entirety, describes a stable phosphate esterlinkage for permanent attachment of oligonucleotides to a glass surface.Mandenius et al., Anal. Biochem. 157; 283 (1986), incorporated byreference herein in its entirety, teaches that the hydroxyalkyl groupresembles the 5′-hydroxyl of oligonucleotides and provides a stableanchor on which to initiate solid phase synthesis. Other suchbinding/release technologies are also known or may become available andare thus contemplated for use in the practice of the present invention.

The efficiency of oligonucleotide synthesis can be greatly enhanced byemploying nucleotide building blocks that are a combination of monomersand multimers. Examples of nucleotide building blocks includenucleotides, analogues or derivatives thereof containing reactive,blocking or other groups rendering the nucleotide building blocksuitable for reaction to form oligonucleotides. Thus, in a particularaspect of the forgoing synthesis embodiment, there are provided methodsfor oligonucleotide synthesis in which each source pool contains analiquot comprising one member from the group consisting of anoligonucleotide of 10 or more nucleic acid bases, a dimericoligonucleotide (e.g., all possible combinations of an oligonucleotidecomprising two nucleotide bases), a trimeric oligonucleotide (e.g., allpossible combinations of an oligonucleotide comprising three nucleotidebases), a tetrameric oligonucleotide (e.g., all possible combinations ofan oligonucleotide comprising four nucleotide bases), and a pentamericoligonucleotide (e.g., all possible combinations of an oligonucleotidecomprising five nucleotide bases). As used herein a nucleotide base isselected from the group consisting of adenine, cytosine, guanine andthymine (or uracil). A complete set of all possible nucleotidecombinations equals 1,024 possible pentamers combinations, 256tetramers; combinations, 64 trimers combinations, 16 dimerscombinations, and 4 monomers, which can easily be placed into anindustry standard 1,536 well plate, as only 1,364 individual wells arerequired of the total 1,536 available. A computer can determine the mostefficient synthesis scheme for a desired product oligonucleotide byoptimally selecting building blocks from the source fluid wellscontaining the oligonucleotide material comprising the monomer throughpentamer oligonucleotides, and thereby minimize the number of stepsrequired to synthesize the desired product oligonucleotide. For example,the present invention allows for the synthesis of 1.0995×1012 possible20-mer oligonucleotide combinations with only 4 couplings using anycombination of the pentamer source fluid materials. Similarly, 12couplings of any combination of the pentamer source fluid materials willgive rise to 1.329×10³⁶ possible 60-mer oligonucleotide combinations.Thus, oligonucleotide synthesis can be automated and conducted withgreater efficiency than if the synthesis were conducted by the stepwiseaddition of single nucleotides only. Other extended sequence iterativesynthesis reactions may also be performed by the methods of theinvention.

In a further embodiment of the present invention, there are providedmethods for determining or confirming the nucleotide sequence of an“unknown” polynucleotide. The polynucleotide may be labeled byconventional methods (e.g., fluorescent, magnetic or nuclear) and thencontacted with target oligonucleotides of known sequence that havepreviously been bound to an array of sites on the target using themethods of the invention (i.e., ejection of the known oligonucleotidefrom a source pool to a desired target zone on the target array).Indeed, the target oligonucleotides may be synthesized in situ on thetarget array using methods described herein. Following contacting of the“unknown” polynucleotide with the target array of oligonucleotides, thetarget array is washed at the appropriate stringency and the presenceand location of hybridized-labeled polynucleotide is determined usingscanning analyzers or the like. Since the sequence of the targetoligonucleotide at each position of the target array is known, thisembodiment of the invention provides for the unambiguous determinationof the nucleotide sequence of the selected polynucleotide.

In performing the methods of the invention, the volume of each of thesource pools is depleted as material is ejected from them. Thus, it isdesirable to monitor the volume or level of each source pool to ensurefluid is available. The volume of level of source fluid is alsoimportant because the impinging acoustic wave or beam will ejectdroplets from the surface of the source pool most efficiently if thebeam is focused as nearly as possible on the surface of the pool. Thus,by monitoring the volume or level of the source pool, the focus of theacoustic wave or beam can be adjusted (e.g., by adjusting the distancebetween the acoustic deposition emitter the source fluid containmentstructure).

Accordingly, in a further embodiment the invention provides a method fordetecting the amount of source fluid remaining in a source pool. Fluidvolume or level detection may be performed by a variety of methodsincluding direct visual/optical inspection, indirect measurement, andthe like. In one aspect of this embodiment, detecting is performed byoptically observing a change in the source fluid volume or level as aresult of ejecting said droplet from said pool. In this aspect, opticalobservation may be performed by an optical detector coupled to acomputer, wherein the computer computes a change in volume or levelbased on signals received from the optical detector before ejection of adroplet, and after the ejection of a droplet.

Optical detectors contemplated for use in the practice of the presentinvention may include a camera, a photoelectric cell, and the like. Forexample, a laser or other light source can be directed at the surface ofa source pool and the defraction angle determined by one or morephotoelectric cells coupled to a computer. The angle can thus indicatethe level of fluid in the source pool, and from there, the volume canreadily be computed. Other optical detection methods known to those ofskill in the art or developed in the future may also be employed in thisaspect of the present invention.

In another aspect of the invention, detection of the fluid level (volumeand/or height) may be by observing the acoustic reflection properties ofthe pool of source fluid. For example, by detecting the reflection ofthe acoustic beam employed to eject the droplet from the surface, thevolume can be computed based on empirically determined acousticreflection characteristics. Since the acoustic liquid deposition emitter(e.g., a piezoelectric transducer) design is similar with acousticmeasuring devices the droplet generator's transducer may also be usedfor acoustic depth sensing as a means of pool level or volume feedbackmeasurement. The signal can be processed and the system can then beadjusted to further focus the acoustic wave or beam as the level orvolume changes. In another aspect of this embodiment, a secondarypiezoelectric transducer can be employed to generate the acoustic beamemployed to detect the fluid level. The secondary piezoelectrictransducer may be toroidal and disposed around the perimeter of thepiezoelectric transducer used to eject the droplet of fluid (i.e., theprimary transducer). One example of this embodiment is depicted in FIG.5, which shows two options for deploying a secondary piezoelectrictransducer. For example, a toroidal secondary transducer 65 may bedisposed around the perimeter of the primary piezoelectric transducer60. In another aspect, a non-toroidal secondary transducer 65′ may beemployed to generate the acoustic wave used to gauge fluid level. Otherdeployments of the second piezoelectric transducer may also be employedin the practice of the present invention.

Any of the forgoing embodiments for detecting fluid level may beemployed in conjunction with a computer that retains and/or manipulatesthe values of the fluid level. In one embodiment of the presentinvention, an example of which is shown in FIG. 6, the liquid surfacereflection of the signal from the secondary piezoelectric element isreceived by a computer 100 which computes the fluid level and sends afluid level value 105 through a comparator algorithm 110 which may thenbe used to send a signal to an actuator 120 that operates to modulateone or more parameters (e.g., energy used to fire the piezoelectricelement, distance of the piezoelectric element and/or lens from thesurface of the source pool, and the like) in order to achieve thedesired focus and energy of the acoustic wave. If desired, a returnsignal 115 of one or more values such as emitter position or the like,can be returned to the comparator algorithm for further evaluation.

In addition, a computer can be used to control any number ofcontrollable parameters including, for example, a stage locationrelative to the deposition emitter (e.g., piezoelectric transducer),frequency, voltage and duration of an energy source used to excite theacoustic liquid deposition emitter.

The various techniques, methods, and aspects of the invention describedabove can be implemented in part or in whole using computer-basedsystems and methods. Additionally, computer-based systems and methodscan be used to augment or enhance the functionality described above,increase the speed at which the functions can be performed, and provideadditional features and aspects as a part of or in addition to those ofthe invention described elsewhere in this document. Variouscomputer-based systems, methods and implementations in accordance withthe above-described technology are presented below.

A computer useful in the invention can be a processor-based systemincluding a main memory, preferably random access memory (RAM), and canalso include a secondary memory. The secondary memory can include, forexample, a hard disk drive and/or a removable storage drive,representing a floppy disk drive, a magnetic tape drive, an optical diskdrive, etc. The removable storage drive reads from and/or writes to aremovable storage medium. Removable storage media represents a floppydisk magnetic tape, optical disk, etc., which is read by and written toby removable storage drive. As will be appreciated, the removablestorage media includes a computer usable storage medium having storedtherein computer software and/or data. The stored data and/or softwarecan include instructions to cause the computer to control a movablestage, frequency, voltage and duration of an energy source used toexcite the acoustic liquid deposition emitter, for example.

In alternative embodiments, secondary memory may include other similarmeans for allowing computer programs or other instructions to be loadedinto a computer system. Such means can include, for example, a removablestorage unit and an interface. Examples of such can include a programcartridge and cartridge interface (such as the found in video gamedevices), a movable memory chip (such as an EPROM, or PROM) andassociated socket, and other removable storage units and interfaceswhich allow software and data to be transferred from the removablestorage unit to the computer system.

The computer system can also include a communications interface.Communications interfaces allow software and data to be transferredbetween computer system and external devices. Examples of communicationsinterfaces can include a modem, a network interface (such as, forexample, an Ethernet card), a communications port, a PCMCIA slot andcard, etc. Software and data transferred via a communications interfaceare in the form of signals which can be electronic, electromagnetic,optical or other signals capable of being received by a communicationsinterface. These signals are provided to the communications interfacevia a channel capable of carrying signals and can be implemented using awireless medium, wire or cable, fiber optics or other communicationsmedium. Some examples of a channel can include a phone line, a cellularphone link, a RIF link, a network interface, and other communicationschannels. The computer interface or communications ports can be used toreceive instructions or to cause an apparatus operably connected to thecomputer to perform a particular function.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples are intended to illustratebut not to limit the invention in any manner, shape, or form, eitherexplicitly or implicitly. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

While the invention has been described in detail with reference tocertain d embodiments thereof, it will be understood that modificationsand variations in the spirit and scope of that which is described andclaimed.

1-57. (canceled)
 58. A method for generating an array of molecularmoieties on a porous substrate surface divided into a plurality ofdiscrete surface sites, the method comprising applying focused acousticenergy to each of a plurality of a reservoirs each containing amolecular moiety in a fluid, wherein the focused acoustic energy isapplied using an acoustic ejector comprised of an acoustic radiationgenerator and a focusing means in a manner effective to eject a dropletfrom each reservoir toward the substrate surface such that the molecularmoiety in each droplet attaches to a localized region within a differentsurface site.
 59. The method of claim 58, wherein each molecular moietyis different.
 60. The method of claim 59, wherein a droplet is ejectedtoward each surface site, such that every surface site has a molecularmoiety attached thereto.
 61. The method of claim 60, wherein eachmolecular moiety is different.
 62. The method of claim 58, wherein themolecular moieties are biomolecules.
 63. The method of claim 62, whereinthe biomolecules are nucleotidic.
 64. The method of claim 63, whereinthe biomolecules are oligonucleotides.
 65. The method of claim 64,wherein the biomolecules are nucleotidic monomers, and the methodfurther comprises stepwise synthesis of an oligonucleotide within eachsurface site by repeated deposition of individual nucleotidic monomersat each site using focused acoustic energy.
 66. The method of claim 62,wherein the biomolecules are peptidic.
 67. The method of claim 60,wherein the porous substrate surface is comprised of more than onethousand discrete surface sites.
 68. The method of claim 60, wherein theporous substrate surface is comprised of thousands of discrete surfacesites.
 69. The method of claim 60, wherein the porous substrate surfaceis comprised of many thousands of discrete surface sites.
 70. The methodof claim 60, wherein the porous substrate surface is comprised of morethan one thousand discrete surface sites on a microscope slide.
 71. Themethod of claim 60, wherein the porous substrate surface is comprised ofthousands of discrete surface sites on a microscope slide.
 72. Themethod of claim 60, wherein the porous substrate surface is comprised ofmany thousands of discrete surface sites on a microscope slide.
 73. Amolecular array comprised of a plurality of different molecular moietieson a porous substrate surface divided into a plurality of discretesurface sites, each site containing one molecular moiety attached to thesubstrate surface in a localized region within the site, wherein morethan one thousand different sites are present.
 74. The molecular arrayof claim 73, wherein the more than one thousand different sites arepresent on a microscope slide.
 75. The molecular array of claim 73,wherein thousands of different sites are present.
 76. The moleculararray of claim 73, wherein the thousands of different sites are presenton a microscope slide.
 77. The molecular array of claim 73, wherein manythousands of different sites are present.
 78. The molecular array ofclaim 73, wherein the many thousands of different sites are present on amicroscope slide.
 79. The molecular array of claim 73, wherein eachmolecular moiety is different.
 80. The molecular array of claim 73,wherein the molecular moieties are biomolecules.
 81. The molecular arrayof claim 80, wherein the biomolecules are oligonucleotides.
 82. Themolecular array of claim 81, wherein the biomolecules are peptidic.